Intracellular transport is ubiquitous in animal cells and has fundamental importance for diverse biological phenomena, such as secretion, neuronal signaling, organization of endomembranes, and mitosis. The driving force for intracellular transport is provided by molecular motors bound to the surface of cargo organelles and moving along microtubules (MTs) and actin filaments (AFs). Switching between these two types of transport must be precisely regulated for the delivery of organelles to specific regions of the cytoplasm. However, the mechanisms of such regulation remain a mystery. Here, we propose to answer the question about the regulation of switching between MTs and AFs using Xenopus melanophores as an experimental system. These cells rapidly redistribute in the cytoplasm thousands of membrane-bounded pigment granules, which aggregate in the cell center or disperse throughout the cytoplasm by means of pigment granule-bound molecular motors that use both MT and AF tracks. Switching of pigment granules between MTs and AFs is controlled by the levels of the second messenger cAMP. Background data by the principal investigator suggest that switching between the two types of tracks is based on a continuous tug-of-war between transport systems. The outcome of the tug-of-war is decided by the relative activities of MT-based and AF-based molecular motors simultaneously bound to the same pigment granule. Preliminary data also indicate that besides molecular motors, organelle transport and its switching between MT and AF tracks involves additional proteins that regulate docking of organelles to the destination track. This proposal will use molecular, cellular, and biochemical approaches to test the hypothesis that aggregation and dispersion signals regulate organelle docking and activities of granule-bound molecular motors through interconnected mechanisms that generate distinct kinetics of transferring pigment granules between MTs and AFs during aggregation and dispersion. To examine these regulatory mechanisms, docking molecules will be identified, and the regulation of their binding to transport tracks and pigment granules will be elucidated. The role of phosphorylation of subunits of molecular motors in regulation of their activities will be also determined.